Project Summary/Abstract Melanoma has the capacity to metastasize early and its course is rarely impacted by medical intervention. Because of the pronounced difference in survival between localized and metastatic disease, it is imperative to diagnose melanoma in its earliest form; however, even expert pathologists can have difficulty distinguishing melanomas from benign nevi (moles), especially atypical nevi. DNA methylation holds promise as a tool, in conjunction with histopathology, for enhancing melanoma diagnosis by providing molecular information. High- throughput DNA-methylation array profiling has the potential for discovery of candidate DNA-methylation sites useful for diagnosis but must be valid on formalin-fixed paraffin-embedded (FFPE) tissue, which is typically the only diagnostic material available for melanocytic lesions. In an R21 phase, we demonstrated technical feasibility of DNA-methylation profiling using FFPE tissues and identified a proof-of-principle signature for discriminating melanomas from nevi. However, many challenges remain in the application of this technology to FFPE tissues, including the ability of the assay to handle small specimens and methods for verifying percent tumor. Our long-term goal is to develop a practical clinical assay for molecular diagnosis of melanoma at the earliest possible stage, while avoiding false-positives and minimizing the overall cost of diagnosis. The objectives in this application are to identify internal markers to assess percent tumor, determine valid conditions for high-throughput DNA-methylation profiling of small FFPE tissues; and confirm DNA-methylation differences using additional assays. The central hypothesis of our proposal is that high-throughput DNA- methylation array profiling of FFPE tissues can be further developed to handle small samples and internal standards can be identified to assess percent tumor. The rationale for the proposed work is that allowing the majority of samples to be profiled will decrease bias in selection of candidate DNA-methylation differences, while the internal standards will exclude samples that will not give valid results. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify candidate DNA-methylation differences that distinguish melanocytic (nevus or melanoma) vs. non-melanocytic (surrounding skin or lymphocytic infiltrate) cells for use as internal quality control standards to quantify sample percent tumor; 2) Identify valid conditions for high-throughput DNA-methylation array profiling of small-sized FFPE melanocytic tissues; and 3) Confirm candidate DNA-methylation differences from high-throughput profiling using more quantitative assays. The approach is innovative because it couples emerging high-throughput DNA- methylation technology to a biospecimen repository and FFPE melanocytic specimens, as they are typically prepared in hospital and community-based dermatologic practices. The proposed research is significant because valid high-throughput DNA-methylation profiling of the majority of FFPE specimens along with internal quality control measures will open new diagnostic opportunities for melanoma and other malignancies.